Part 3 (final exam)The cytoskeleton- assembly and dynamic structure- Functions (movement and structure)o 1. Maintaining and directing cell structureo 2. Intracellular support Organelle movement Vesicle movemento 3. Spatial organization of cello 4. Contractility and Mobility Actin/myosin contraction Movement of cilia and flagella Chromosome movement Cell migration- amoeboid movement- Studying the cytoskeletono Fluorescence Microscopy- to see the fixed structure Immunofluorescence : use of antibodies against cytoskeleton proteins- Stain a fixed section of the cell with antibodieso The ones tagged with red proteins are directed against actino Green- tubulino Blue- nucleus Use of fluorescently-tagged drugs that bind to cytoskeleton proteins - E.g. phalloidin- binds to actin filamentso Video-enhanced light microscopy- can detect presence of tubules and filament bundles, and follow movement of vesicles in living cells (but can’t resolve details of structure)o Electron Microscopy- especially of freeze-etched surfaceso Genetic engineering- to study the FUNCTION of the cytoskeleton The development of genetically engineered organisms or cell lines that lack acytoskeletal or motor protein (knockout mutants) has been enormously productive- Mouse embryos that lack a motor protein called cytoplasmic dynein fail to develop beyond 8 days sue to dispersion and fragmentation of the Golgi complex, suggesting a role for dynein in maintaining the structural integrity of the Golgio Golgi breaks into vesicles in the absence of dynein1 Cell lines or organisms can be engineered to over-express an artificially-constructed “dominant negative” form of a cytoskeletal protein.- “Dominant-negative” proteins are non-functional mutant proteins that compete with the native protein and inhibit its functiono Interferes with the function of a normal proteino If you are able to develop and express this protein, you can knock out the function of the protein you are targeting withoutknocking out the gene of the protein you are targeting- Cells expressing the dominant negative protein show defects in the specific functions of the protein similar to those of knockout mutants Cells that are engineered to express “small interfering” RNA sequences (siRNA’s) targeted to be complementary to the mRNA’s of specific cytoskeletal proteins may also show defect in functions associated with those proteins- The siRNA binds to the specific mRNA that has the complementary sequence, promotes its destruction and silences the translation of the protein that the mRNA specifically codes for- siRNAs can trigger the destruction of mRNA- Cytoskeleton is formed from:o 1. Microtubuleso 2. Intermediate filamentso 3. Microfilaments- Cytoskeleton can be dynamic (changing) or stable:o Dividing fibroblastso Stable- Epithelial cells in the gut- Mictotubuleso Large arrays of rigid microtubule bundles help maintain the overall shape of many cell types2o Maintain the internal organization of cells Treatment with microtubule-disorganizing drugs can seriously disrupt organelle distribution- i.e. the Golgi is dispersed throughout the cell, instead of being localized just outside the nucleus- In plant cells, microtubule bundles at the edge of the cytoplasm direct cellulose deposition in the neighboring cell wall by influencing the positioning of cellulose-synthesizing enzymeso Important in maintaining the shape of very elongated cell extensions or processes i.e. Axons of nerve cells- treatment with microtubule-disorganizing drugs causes collapse of growing axons in the developing nervous systemo Form the: Mitotic spindles of dividing cells The core of cilia and flagella A network of rigid tubules that radiate through the cytoplasm of all eukaryotecellso Formed from alpha and beta tubulin Globular proteins- approx. 50kDa M.W.o Structure: A rigid tube, whose wall is made from approx. 13 protofilaments, composed of alternating alpha and beta tubulins Approx. 25 nm in diameter and up to a micron or more in length The filament is polarized, with a (+) and a (-) end- (+) end is composed of a row of beta tubulins; grows more rapidly- (-) end is composed of a row of alpha tubulinso Proteins that bind to microtubules: 1. Motor proteins- kinesins and dyneins 2. MAPS (microtubule associated proteins)- proteins that cross-link MT’s or regulate their assembly - MAP2- its long projecting arm creates bundles of widely-spaced microtubule arrays- Binding of MAPS to microtubules is often regulated by their phosphorylation3o The tau protein- a protein that regulates microtubule assembly in neurons Overphosphorylation of tau may be involved in creating the tangled neuronal structures observed in Alzheimer’s Diseasepatientso Drugs that affect microtubules: Nocodazole - prevents microtubule assembly Taxol - prevents microtubule disassembly Both of these drugs are anti-mitotic (prevent cell division) since they disrupt the assembly/disassembly of microtubules that are needed to allow chromosomes to separate Taxol is used for chemotherapy since it will prevent division of tumor cells…but also prevents mitosis in other cells (hair follicleshair loss)o Assembly: Assembly of microtubules both in vitro and in vivo begins with the creation of dimers between alpha and beta tubulin- Dimerization requires that a GTP molecule is bound to the beta tubulin. The dimers then associate into microtubule fragments and then elongate to form microtubules- The GTP is hydrolyzed some time after the dimer is incorporated into a microtubule, leaving GDP’s still attached to tubulin subunits in the assembling microtubule Tubulin assembly can be achieved in cell homogenates in a test tube (in vitro- dead organisms)- Needs alpha tubulin, beta tubulin, and the addition of Mg2+, GTP, EGTA (binds Ca2+- an inhibitor of assembly)- Assembly/disassembly can be produced by raising/lowering temperature- Can be sped up by adding existing microtubule fragments- nucleation occurs on the fragmentso Thus, nucleation is the rate-limiting step of assembly During periods of rapid growth of the (+) end, most tubulins present still haveGTP attached, creating a GTP “cap” with a different structure from the (-) end, where GDP predominates- This cap stabilizes the (+) end4- “Dynamic instability” of the microtubule is the ebb and flow of microtubule length during a cell’s
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